Abstract:

APPLICANT?S DESCRIPTION: Gene therapy will allow for the treatment of both
acquired and genetic diseases at the most fundamental level by introduction of
therapeutic genes. Despite advances in delivery of foreign genes, achieving
high and stable levels of expression has been problematic. The use of plasmid
DNA for gene therapy has many advantages, including safety, accommodation of
large genes, ease of production, and low cost. Plasmid DNA is lost, however, as
cells divide. Viral integration of a transgene into the host genome prevents
loss of the gene. Integration is hypothesized to be responsible for the
long-term expression of some transgenes delivered through viral vectors.
Disadvantages of viral vectors include safety concerns, cost, and difficulties
in production. In order to achieve nonviral integration of a transgene and then
expression at therapeutically useful levels, we propose to deliver the gene in
pre-formed synaptic complexes of hyperactive Tn5 transposase dimers bound to
DNA elements flanking the transgene to be integrated. This integration requires
no specific sequence in the target DNA (the genome). During Phase I, we will
verify that these synaptic complexes can be delivered into mammalian cells in
culture and that the bacterial transposase can indeed effect integration in
mammalian cells. In Phase II, the use of pre-formed Tn5 transposase-DNA
complexes will be optimized and incorporated into gene therapy approaches such
as the transplantation of cells genetically modified ex vivo (e.g.,
fibroblasts, keratinocytes, and myoblasts) or the direct gene transfer into
cells in vivo (e.g., hepatocytes). These studies will form the basis for the
commercial development of Tn5 transposase-mediated integration of transgenes
into mammalian cells in vitro as a research tool, and for gene therapy ex vivo
and in vivo.
PROPOSED COMMERCIAL APPLICATION:
A gene integration system could have immediate application for ex vivo gene therapy (e.g.,
transplantation of genetically modified fibroblasts, keratinocytes, hemopoietic stem cells, and
myoblasts). Mirus will immediately commercialize (on the basis of the Phase I studies) reagents
for enabling the more efficient integration of cells in culture (in conjunction with our line of
transfection reagents) and cells in vivo (on the basis of Phase II studies). Its commercial
development will be accomplished by initiation of pre-clinical trials using model disorders
such as hemophilia and licensing to larger pharmaceutical and biotechnology companies.